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LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.033, PP. 214-219

Keywords: 细菌双杂交,诱饵载体,基因组文库,早期基因,甲型副伤寒沙门氏菌

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Abstract:

构建细菌双杂交系统中的诱饵载体pKT25-gp22和甲型副伤寒沙门氏菌基因文库以便后续的筛选实验。PCR扩增获得gp22基因,插入pKT25构成诱饵质粒pKT25-gp22。提取甲型副伤寒沙门氏菌基因组DNA,经Sau3AⅠ部分酶切后连接到pUT18C质粒的BamHⅠ位点,获得基因组DNA表达文库。用化转的方法将诱饵质粒与pUT18C共转入BTH101,检测诱饵质粒自激活作用,并检测诱饵蛋白对宿主菌的毒性。将文库质粒电转至JM109感受态,PCR鉴定文库的多样性。诱饵载体pKT25-gp22无自激活报告基因的能力且对细菌的生长无毒性。所构建的副甲基因组文库覆盖基因组达8倍,满足文库筛选需要。成功构建细菌双杂交系统中诱饵载体pKT25-gp22,筛选文库质量良好,可应用于细菌双杂交实验。

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